Improvement of spinal non-viral IL-10 gene delivery by D-mannose as a transgene adjuvant to control chronic neuropathic pain.

BACKGROUND: Peri-spinal subarachnoid (intrathecal; i.t.) injection of non-viral naked plasmid DNA encoding the anti-inflammatory cytokine, IL-10 (pDNA-IL-10) suppresses chronic neuropathic pain in animal models. However, two sequential i.t. pDNA injections are required within a discrete 5 to 72-hour period for prolonged efficacy. Previous reports identified phagocytic immune cells present in the peri-spinal milieu surrounding the i.t injection site that may play a role in transgene uptake resulting in subsequent IL-10 transgene expression. METHODS: In the present study, we aimed to examine whether factors known to induce pro-phagocytic anti-inflammatory properties of immune cells improve i.t. IL-10 transgene uptake using reduced naked pDNA-IL-10 doses previously determined ineffective. Both the synthetic glucocorticoid, dexamethasone, and the hexose sugar, D-mannose, were factors examined that could optimize i.t. pDNA-IL-10 uptake leading to enduring suppression of neuropathic pain as assessed by light touch sensitivity of the rat hindpaw (allodynia). RESULTS: Compared to dexamethasone, i.t. mannose pretreatment significantly and dose-dependently prolonged pDNA-IL-10 pain suppressive effects, reduced spinal IL-1ß and enhanced spinal and dorsal root ganglia IL-10 immunoreactivity. Macrophages exposed to D-mannose revealed reduced proinflammatory TNF-α, IL-1ß, and nitric oxide, and increased IL-10 protein release, while IL-4 revealed no improvement in transgene uptake. Separately, D-mannose dramatically increased pDNA-derived IL-10 protein release in culture supernatants. Lastly, a single i.t. co-injection of mannose with a 25-fold lower pDNA-IL-10 dose produced prolonged pain suppression in neuropathic rats. CONCLUSIONS: Peri-spinal treatment with D-mannose may optimize naked pDNA-IL-10 transgene uptake for suppression of allodynia, and is a novel approach to tune spinal immune cells toward pro-phagocytic phenotype for improved non-viral gene therapy.

Chagas disease: a proposal for testing policy for solid-organ transplant in the United States.

Chagas disease is caused by the parasite Trypanosoma cruzi. The disease is difficult to detect because of the asymptomatic pathogenesis after infection. Chagas disease is endemic throughout much of Mexico, Central America, and South America, but human migration patterns are bringing the pathogen to the United States. The disease currently affects 16 to 18 million people with approximately 50 000 deaths annually in these countries. In the United States, national screening of the blood supply was instituted in early 2007, and more than 1000 donors with T cruzi infection have been identified within the past 3 years of testing. It was observed that out of the 58 organ procurement organizations in the United States, only 4 required mandatory testing of every donor for Chagas disease. It was estimated that as of 2009, approximately 409 000 residents are living with Chagas disease, and in a 22-year span, approximately 300 patients may have contracted Chagas disease through transplant. Proposed solutions to the current testing method include automatic testing based on the medical social history questionnaire, testing of all recipients for Chagas disease, testing all persons of Latin descent, or testing of all organ donors.

Treatment rationale and study design for a randomized, double-blind, placebo-controlled phase II study evaluating onartuzumab (MetMAb) in combination with bevacizumab plus mFOLFOX-6 in patients with previously untreated metastatic colorectal cancer.

BACKGROUND: Dysregulation of the hepatocyte growth factor (HGF)/MET pathway is associated with poor prognosis, more aggressive biological characteristics of the tumor, and shortened survival in patients with metastatic colorectal cancer (mCRC). Onartuzumab (MetMAb) is a recombinant humanized monovalent monoclonal antibody directed against MET. We present the treatment rationale and protocol for an ongoing randomized multicenter placebo-controlled phase II study designed to evaluate the efficacy and safety of MetMAb combined with bevacizumab and mFOLFOX-6 (5-fluoruracil, leucovorin, and oxaliplatin). PATIENTS AND METHODS: Eligible patients with previously untreated mCRC are randomized 1:1 to either mFOLFOX-6 combined with bevacizumab and placebo followed by 5-fluorouracil/leucovorin plus bevacizumab and placebo or mFOLFOX6, bevacizumab plus MetMAb followed by 5 FU/LV, bevacizumab, and MetMAb. The primary end point of this study is progression-free survival (PFS) in the intent-to-treat (ITT) population. Secondary end points include overall survival (OS), objective response rate, and safety. Subanalyses will be performed to evaluate the effect of MET receptor expression on study primary and secondary end points. Correlative studies will be performed on tissue- and blood-derived biomarkers related to both HGF/MET signaling and other associated pathway markers.

The inhibitory activity of typified propolis against Enterococcus species.

Propolis, a natural bee product widely used for its antimicrobial activity, was tested against isolates of Enterococcus from humans, pig-tailed macaques, isolates of refractory endodontic treatment cases, and isolates from Lactobacillus-containing food supplements. Typification of the propolis was performed by high-performance liquid chromatography (HPLC) by which prenylated compounds, cinnamic acid derivatives, and flavonoids were detected as the main constituents. Minimum inhibitory concentrations (MIC) were determined using the agar dilution method. All human and animal Enterococcus isolates demonstrated MIC values of 1600 microg/mL. Enterococcal species of human and animal origin were inhibited by propolis. Particularly, human isolates of E. faecium and E. faecalis of refractory endodontic treatment cases were susceptible to propolis of Brazilian origin.

Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia.

During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB(2)R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen-activated protein kinase, interleukin-1ß (IL-1ß), the anti-inflammatory cytokine, interleukin-10 (IL-10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1ß, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti-inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.

Intrathecal cannabilactone CB(2)R agonist, AM1710, controls pathological pain and restores basal cytokine levels.

Spinal glial and proinflammatory cytokine actions are strongly implicated in pathological pain. Spinal administration of the anti-inflammatory cytokine interleukin (IL)-10 abolishes pathological pain and suppresses proinflammatory IL-1ß and tumor necrosis factor alpha (TNF-α). Drugs that bind the cannabinoid type-2 receptor (CB(2)R) expressed on spinal glia reduce mechanical hypersensitivity. To better understand the CB(2)R-related anti-inflammatory profile of key anatomical nociceptive regions, we assessed mechanical hypersensitivity and protein profiles following intrathecal application of the cannabilactone CB(2)R agonist, AM1710, in 2 animal models; unilateral sciatic nerve chronic constriction injury (CCI), and spinal application of human immunodeficiency virus-1 glycoprotein 120 (gp120), a model of peri-spinal immune activation. In CCI animals, lumbar dorsal spinal cord and corresponding dorsal root ganglia (DRG) were evaluated by immunohistochemistry for expression of IL-10, IL-1ß, phosphorylated p38-mitogen-activated-kinase (p-p38MAPK), a pathway associated with proinflammatory cytokine production, glial cell markers, and degradative endocannabinoid enzymes, including monoacylglycerol lipase (MAGL). AM1710 reversed bilateral mechanical hypersensitivity. CCI revealed decreased IL-10 expression in dorsal spinal cord and DRG, while AM1710 resulted in increased IL-10, comparable to controls. Adjacent DRG and spinal sections revealed increased IL-1ß, p-p38MAPK, glial markers, and/or MAGL expression, while AM1710 suppressed all but spinal p-p38MAPK and microglial activation. In spinal gp120 animals, AM1710 prevented bilateral mechanical hypersensitivity. For comparison to immunohistochemistry, IL-1ß and TNF-α protein quantification from lumbar spinal and DRG homogenates was determined, and revealed increased DRG IL-1ß protein levels from gp120, that was robustly prevented by AM1710 pretreatment. Cannabilactone CB(2)R agonists are emerging as anti-inflammatory agents with pain therapeutic implications.

Dasatinib in previously treated metastatic colorectal cancer: a phase II trial of the University of Chicago Phase II Consortium.

BACKGROUND: Treatment options for metastatic colorectal cancer (CRC) are limited after a fluoropyrimidine, oxaliplatin and irinotecan; novel agents need to be explored in this setting. Dasatinib, an oral inhibitor of Src family kinases, inhibits proliferation in CRC cell lines and has antitumor activity in CRC xenograft models. PATIENTS AND METHODS: We conducted a multi-center phase II trial of dasatinib in unresectable, previously-treated metastatic CRC patients. No more than 2 prior chemotherapy regimens were permitted, which must have contained a fluoropyrimidine, oxaliplatin and irinotecan. The primary endpoint was progression-free survival (PFS) at 4 months. The Simon two-stage design required that at least 5 of the first 19 patients be progression-free at 4 months to expand to a second stage. RESULTS: Nineteen patients enrolled at 9 centers. The study was terminated after the first stage due to lack of efficacy. There were no objective responses; 1 patient (5%) had stable disease for 7.3 months. The PFS rate at 4 months was 5.3% (90% CI: 0.3, 22.6). Median PFS was 1.6 months (90% CI: 1.4, 1.8). Median overall survival was 5.1 months (90% CI: 2.4, 6.3). Grade 3/4 toxicities included fatigue in 16% of patients, and anemia, anorexia, nausea/vomiting and dyspnea in 11%. CONCLUSION: Dasatinib is inactive as a single agent in previously treated metastatic CRC patients.

Gemcitabine plus sorafenib in patients with advanced pancreatic cancer: a phase II trial of the University of Chicago Phase II Consortium.

BACKGROUND: Sorafenib, an inhibitor of B-raf, VEGFR2, and PDGFR-ß, has activity against pancreatic cancer in preclinical models. In a phase I trial of gemcitabine plus sorafenib, 57% of pancreatic cancer patients achieved stable disease. PATIENTS AND METHODS: We conducted a multi-center phase II trial of sorafenib plus gemcitabine in chemo-naïve patients with histologically-confirmed, advanced pancreatic cancer. Patients received sorafenib 400 mg twice daily and gemcitabine 1,000 mg/m(2) on days 1, 8 and 15 of a 28 day cycle. RESULTS: Seventeen patients enrolled at 4 centers; 13 were evaluable for response. There were no objective responses; 18% had stable disease. Median overall survival was 4.0 months (95% CI: 3.4, 5.9); median progression-free survival was 3.2 months (95% CI: 1.6, 3.6). Grade 3/4 toxicities included thrombosis in 18% of patients, dehydration or hand-foot syndrome in 12%, and hypertension or gastrointestinal bleeding in 6%. CONCLUSION: Gemcitabine plus sorafenib is inactive in advanced pancreatic cancer.

Cancer in long-term care.

This article describes the range of cancer patients in longterm care and provides a framework for clinical decision making. The benefits and burdens of providing standard therapy to a vulnerable population are discussed. To give more specific guidelines for advocates of treatment, skeptics, and others, the authors present best estimates of the current burden of cancer in the long-term care population and current screening guidelines that apply to the elderly under long-term care. Experience-based suggestions are offered for oncologists and clinicians involved in long-term care to help them respond to patient and family concerns about limitations of cancer care.

Communication about cancer near the end of life.

Cancer communication near the end of life has a growing evidence base, and requires clinicians to draw on a distinct set of communication skills. Patients with advanced and incurable cancers are dealing with the emotional impact of a life-limiting illness, treatment decisions that are complex and frequently involve consideration of clinical trials, and the challenges of sustaining hope while also having realistic goals. In this review, the authors sought to provide a guide to important evidence about communication for patients with advanced cancer regarding communication at diagnosis, discussing prognosis, decision making about palliative anticancer therapy and phase 1 trials, advance care planning, transitions in focus from anticancer to palliative care, and preparing patients and families for dying and death.

Effect of Waterlase laser retrograde root-end cavity preparation on the integrity of root apices of extracted teeth as demonstrated by light microscopy.

Most endodontists use ultrasonic instruments for retrograde root-end cavity preparations even though they have been found to produce cracks. In this laboratory study, thirty-six randomly chosen roots had root-end cavity preparations made with the Waterlase laser and only one questionable intra-canal crack was found. It was concluded that the Waterlase laser when used to make endodontic root-end cavity preparations produces either no cracks, or a very low percentage (2.8%) of cracks.

Effect of iterative restoration on the detection of artificially induced vertical radicular fractures by Tuned Aperture Computed Tomography.

OBJECTIVE: The purpose of this study was to evaluate the effect of the number of iterative restorations (IR) on the diagnostic accuracy of Tuned Aperture Computed Tomography images (TACT) for detection of induced vertical/oblique root fractures in mandibular teeth. STUDY DESIGN: Fractures were induced in 28 single-rooted, endodontically treated cadaver mandibular teeth while another 26 such teeth served as controls. TACT slices reconstructed by using 9 arbitrary projections (angular disparity of 9 degrees -20 degrees) were iteratively restored 1, 2, and 3 times. Eight observers scored their diagnoses by using a confidence rating scale. Ground truth was available from direct examination with transillumination, following extraction. RESULTS: Mean areas under the representative ROC curves for the 3 operations were 0.96 (3 IRs), 0.87 (2 IRs), and 0.86 (1 IRs). Analysis of variance demonstrated significant differences between 3 and 1 to 2 IRs (P <.01), as well as between observers (P <.01). CONCLUSION: Diagnostic accuracy of TACT for vertical/oblique root fracture detection improved after 3 IRs.

Tissue inhibitor of metalloproteinase-3 is associated with neuronal death in reperfusion injury.

Programmed cell death occurs in ischemia when cell surface death receptors (DRs) are stimulated by death-inducing ligands (DILs). Matrix metalloproteinases are extracellular matrix-degrading enzymes involved in the shedding of DRs and DILs from the cell surface. Tissue inhibitor of metalloproteinase-3 (TIMP-3), which is bound to the extracellular matrix, has been shown to promote apoptosis in cancer cell lines by inhibiting cell surface sheddases. Since apoptosis is an important mechanism of cell death in ischemia, the authors hypothesized that TIMP-3 would be expressed in ischemic neurons that are undergoing programmed cell death. Spontaneously hypertensive rats had a 90-minute middle cerebral artery occlusion with reperfusion. Transcription of TIMP-3 mRNA was measured by quantitative reverse transcription-polymerase chain reaction at 2, 6, 24 and 48 hours after reperfusion. Western blots were used to measure TIMP-3 protein expression. Spatial distribution and production of TIMP-3 was studied by immunohistochemistry at 3, 24, and 48 hours, 5 days, and 3 weeks. DNA fragmentation in cells dying by necrosis and apoptosis was identified with terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL). After 2 hours of reperfusion, TIMP-3 mRNA increased significantly in both ischemic and nonischemic hemispheres. Western blot analysis confirmed the identity of the TIMP-3, which appeared to be increased on the ischemic side. After 3 hours of reperfusion, TIMP-3 immunostaining was increased in neurons on the ischemic side, and by 24 hours the majority of the ischemic neurons were TIMP-3-positive. Dual-fluorescence staining for TUNEL and TIMP-3 showed that they were coexpressed in many neurons. The results suggest that ischemic neurons express TIMP-3, which may be inhibiting sheddases. The authors propose that TIMP-3 facilitates cell death in ischemic neurons. Further studies are needed to identify the sheddases inhibited by the TIMP-3, and on the effect of inhibition of matrix metalloproteinases on cell death mechanisms.